Construction of a lentiviral vector for RNA interference of PRL-3 gene and its stable expression in SW480 cells / 南方医科大学学报
Journal of Southern Medical University
;
(12): 509-512, 2008.
Artículo
en Chino
| WPRIM
| ID: wpr-280161
ABSTRACT
<p><b>OBJECTIVE</b>To construct a lentiviral vector for RNA interference (RNAi) of PRL-3 gene and establish a human colon carcinoma cell line with PRL-3 gene knock-down.</p><p><b>METHODS</b>The plasmids were constructed expressing two different short hairpin RNAs (shRNA) targeting PRL-3 gene under control by the U6 promoter by lentiviral vector. An entry clone was generated in the pENTR/U6 vector. After identification with sequencing, a recombinant lentiviral vector was obtained using the pENTR/U6 construct and pLenti6/BLOCK-iT TM-DEST vector. The recombinant lentivirus was harvested from 293FT cells co-transfected with optimized ViraPower Packaging Mix and the pLenti6/BLOCK-iT -DEST expression construct. SW480 cells were infected with the recombinant lentivirus and the cells with stable PRL-3 gene knock-down were screened by blasticidin selection. PRL-3 expression was detected using real-time reverse transcription-polymerase chain reaction and Western blotting.</p><p><b>RESULTS</b>A recombinant lentiviral vector expressing shRNAs targeting PRL-3 gene was successfully established and confirmed by DNA sequencing. The recombinant lentivirus were harvested from 293FT cells with a viral titer of 6 x 10(5) pfu/L. Twelve clones of SW480 cells infected with the recombinant lentivirus were selected, and the Clone 1 exhibited significant knock-down of PRL-3 protein expression (72.9%).</p><p><b>CONCLUSION</b>The successful establishment of human colon carcinoma cell line with PRL-3 gene knock-down provide a basis for investigation of the role of PRL-3 gene in the metastasis of human colorectal carcinoma.</p>
Texto completo:
Disponible
Índice:
WPRIM (Pacífico Occidental)
Asunto principal:
Patología
/
Secuencia de Bases
/
Transfección
/
Neoplasias Colorrectales
/
Línea Celular
/
Western Blotting
/
Proteínas Tirosina Fosfatasas
/
Lentivirus
/
Reacción en Cadena de la Polimerasa de Transcriptasa Inversa
/
Interferencia de ARN
Límite:
Humanos
Idioma:
Chino
Revista:
Journal of Southern Medical University
Año:
2008
Tipo del documento:
Artículo
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